TRIM22 induces cellular senescence by targeting PHLPP2 in hepatocellular carcinoma

The ubiquitin-proteasome system is a vital protein degradation system that is involved in various cellular processes, such as cell cycle progression, apoptosis, and differentiation. Dysregulation of this system has been implicated in numerous diseases, including cancer, vascular disease, and neurodegenerative disorders. Induction of cellular senescence in hepatocellular carcinoma (HCC) is a potential anticancer strategy, but the precise role of the ubiquitin-proteasome system in cellular senescence remains unclear. In this study, we show that the E3 ubiquitin ligase, TRIM22, plays a critical role in the cellular senescence of HCC cells. TRIM22 expression is transcriptionally upregulated by p53 in HCC cells experiencing ionizing radiation (IR)-induced senescence. Overexpression of TRIM22 triggers cellular senescence by targeting the AKT phosphatase, PHLPP2. Mechanistically, the SPRY domain of TRIM22 directly associates with the C-terminal domain of PHLPP2, which contains phosphorylation sites that are subject to IKKβ-mediated phosphorylation. The TRIM22-mediated PHLPP2 degradation leads to activation of AKT-p53-p21 signaling, ultimately resulting in cellular senescence. In both human HCC databases and patient specimens, the levels of TRIM22 and PHLPP2 show inverse correlations at the mRNA and protein levels. Collectively, our findings reveal that TRIM22 regulates cancer cell senescence by modulating the proteasomal degradation of PHLPP2 in HCC cells, suggesting that TRIM22 could potentially serve as a therapeutic target for treating cancer.


INTRODUCTION
Hepatocellular carcinoma (HCC) is the primary subtype of liver cancer, accounting for ~90% of all cases.HCC is associated with high rates of tumor recurrence and metastasis after primary hepatic resection, contributing to the most common cause of cancer-related mortality worldwide [1,2].Radiotherapy (RT) is a major modality used in treating HCC, particularly progressive HCC patients with tumors that are not amenable to resection or transplantation, or those with extrahepatic metastasis tumors [2][3][4].However, the efficacy of RT is limited by the endogenous and therapy-induced radioresistance of HCC [5][6][7][8][9].To improve the effectiveness of RT in the treatment of HCC, there is still a need to identify potential therapeutic targets associated with HCC radioresistance.
Cellular senescence is a state of stable cell cycle arrest characterized by changes in morphology, macromolecule compositions, and the acquisition of pro-inflammatory phenotypes.Senescence can be induced by a variety of endogenous and exogenous stressors, such as telomere shortening, mitochondrial dysfunction, DNA damage, and oncogene activation.Moreover, cancer therapies, including chemotherapy, radiotherapy, and targeted therapy, can trigger therapy-induced senescence (TIS).
The induction of cellular senescence is considered as a potential strategy for treating cancer by inducing tumor suppression and immune surveillance [10][11][12].However, senescent cancer cells acquire pro-tumorigenic properties through activation of the senescence-associated secretory phenotype (SASP), which can modulate the tumor microenvironment and increase cancer stemness, invasion, migration, angiogenesis, and immune evasion [7,8].Nonetheless, the induction of cellular senescence remains a promising strategy for combination therapies in cancer treatment.Recent studies have revealed that pro-senescence therapy can increase the vulnerability of tumors to combination treatments, particularly with the use of senolytics and senomorphic agents.This approach provides new avenues for enhancing treatment outcomes and addressing challenges related to senescenceassociated phenotypes [13][14][15].
The ubiquitin-proteasome system (UPS) is an important protein homeostasis mechanism that targets substrates for ubiquitinmediated degradation.This process is mediated by three enzymes: a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a ubiquitin ligase (E3) [16].Dysregulation of E3 ligases is commonly observed in cancer: Changes in E3 ligase expression or activity can affect the progression, development, immune checkpoint regulation, and drug responses of various cancers [17,18].E3 ligases play a crucial role in determining the specificity and selectivity of ubiquitinated substrates, and they can have oncogenic or tumor-suppressive properties depending on their substrates [19][20][21][22][23][24].Understanding the roles of E3 ligases in cancers can provide valuable insights into treating the disease.The activity of oncogenic E3 ligases can be inhibited by small molecules or peptides, such as PROTAC (proteolysis targeting chimeric) or molecular glues.For tumorsuppressive E3 ligases, on the other hand, potential therapeutic strategies include reinstating their expression or activity, exploring synthetic lethality, or targeting downstream oncogenic substrates [17,18].E3 ligases contribute to cellular senescence by suppressing or promoting DNA damage responses and cell cycle arrest [25][26][27][28].The E3 ligases tripartite motif-containing 22 (TRIM22) is a TRIM protein family member and is characterized by the presence of RING, BBox, and Coiled-Coil domain regions at the N-terminus and a SPRY region at the C-terminus.TRIM22 has been implicated in regulating the progression and development of various cancers, including glioblastoma, osteosarcoma, and gastric cancer [29][30][31].However, the potential role of TRIM22 in the cellular senescence of HCC remains completely unexplored.
In this study, we demonstrate that TRIM22 is upregulated by p53 and its overexpression attenuates the AKT phosphatase PHLPP2 via the UPS, resulting in activation of AKT-p53-p21 senescence pathway.Our study suggests that TRIM22 can be a promising target for the cancer treatment.

TRIM22 induces cellular senescence in HCC
To identify E3 ligases that contribute to the therapy-induced senescence of HCC cells, we analyzed an expression profiling array (GSE30240) for IR (ionizing radiation)-induced senescent HepG2 cells.We found that 26 E3 ligases were upregulated (fold change > 2) after IR treatment.When we validated these findings by RT-qPCR, we found that the mRNAs encoding MIB2, PML, TRIM22, TRIM38, HERC6, and TRIM21 were increased by more than 1.5 times in IR-treated HepG2 cells (Fig. 1A).To investigate which E3 ligases play critical roles in the cellular senescence of HCC, we transfected small interfering RNA (siRNA, Si) to knockdown each of the identified E3 ligases in IR-treated HepG2 cells, and monitored typical senescent traits.Only TRIM22 depletion was found to reduce SA-β-Gal positivity (Fig. 1B) and partially rescue the cell number (Fig. 1C) in IR-treated HepG2 cell cultures.We analyzed TRIM22 expression in HCC cell lines with wild-type p53 (Wt p53) (SK-Hep-1, HepG2) or mutant p53 (Mut p53) (SNU449, Huh7, PLC/ PRF/5).TRIM22 was found to be markedly upregulated in HCC cells with Wt p53, but not Mut p53, after IR treatment (Fig. S1A).To verify whether IR induces cell death in HCC cell lines with Wt p53 (SK-Hep-1 and HepG2), we conducted Western blot and caspase-3 activity analyses.The results revealed that the protein levels of p53 and p21 were increased in both SK-Hep-1 and HepG2 after IR treatment (Fig. S1B).However, neither cleaved PARP (C-PARP) nor caspase-3 activity was increased in IR-treated SK-Hep-1 and HepG2 cells, indicating that IR did not induce cell death in SK-Hep-1 and HepG2 cells (Fig. S1B-D).Next, we analyzed TCGA data and found that TRIM22 expression was higher in Wt p53 HCC tissues compared to Mut p53 HCC tissues (Fig. S1E).Furthermore, knockdown of p53 in HepG2 cells resulted in the downregulation of TRIM22 at mRNA and protein levels (Fig. S1F).A chromatin immunoprecipitation (ChIP) assay performed with a p53 antibody in IR-treated HepG2 cells revealed that p53 directly bound to the p53-response element in the intron 1 of TRIM22, and its binding affinity was enhanced upon IR treatment (Fig. S1G).These findings indicated that TRIM22 is positively regulated by Wt p53, which directly binds to the p53-response element in the intron 1 of TRIM22.
Next, we analyzed the correlation between TRIM22 and senescence-associated genes in the TCGA-LIHC database and found that p53, p21, p27, ISG15, and STAT1 were positively correlated with upregulated TRIM22 expression in HCC patient samples (Fig. 1D).These data suggested that TRIM22 might function as an upstream regulator of cellular senescence.To explore the possible biological function of TRIM22 in cellular senescence, we overexpressed TRIM22 in two HCC cell lines: HepG2 and SK-Hep-1 cells.Western blotting analysis confirmed that p53 and p21 are increased in TRIM22-overexpressed HCC cells (Fig. 1E).We observed that TRIM22 overexpression reduced cell proliferation, as indicated by Edu incorporation assay and cell counting (Fig. 1F, G).However, PARP cleavage and caspase-3 activity did not increase in both HepG2 and SK-Hep-1 cells following TRIM22 overexpression (Fig. S2A-D).TRIM22 overexpression decreased cell proliferation without causing cell death (Fig. 1F, G, Fig. S2A-D).Additionally, senescence-associated β-galactosidase (SA-β-Gal) positivity was increased in TRIM22overexpressed HCC cells (Fig. 1H).Taken together, these results suggest that TRIM22 induces cellular senescence by activating the p53-p21 signaling pathway in HCC.

TRIM22 modulates AKT phosphorylation through degradation of PHLPP2
To explore the upstream signaling pathway of p53-p21 in TRIM22mediated HCC senescence, we applied phosphoprotein array analysis.Our results showed that the phosphorylation levels of 278 proteins were changed by TRIM22 overexpression; proteins showing levels with changes of more than 1.2-fold (160 proteins) and less than 0.8-fold (118 proteins) were included for further analysis (Fig. 2A).We found that these phosphorylated proteins were involved in the PI3K-AKT signaling and cellular senescence pathways (Fig. 2B).Increased phosphorylation levels of AKT (T308 and S473) and mTOR (S2448 and S2481) were confirmed by Western blot analysis of TRIM22-overexpressed HepG2 cells (Fig. 2C).
We next investigated whether AKT and p53 are critical as downstream molecules for TRIM22-mediated cellular senescence.Our results revealed that depletion of AKT or p53 decreased p21 accumulation and SA-β-Gal positivity and rescued cell proliferation in TRIM22-overexpressed HepG2 cells (Fig. 2D-F).However, overexpression of TRIM22 in Mut p53 SNU449 cells failed to induce cellular senescence and did not cause cell death (Fig. 2G-I, Fig. S2E, F).These results indicate that AKT and p53 are critical downstream players in TRIM22-mediated cellular senescence.
the protein level, with no change in the mRNA level (Fig. 2K, L).These results suggest that TRIM22 increases AKT phosphorylation by reducing the protein level of the AKT phosphatase, PHLPP2.
Two members of the PHLPP family, PHLPP1 and PHLPP2, function to dephosphorylate AKT [32].To further investigate the involvement of PHLPP1 and/or PHLPP2 in AKT activation for HCC senescence, we depleted PHLPP1 and PHLPP2 by using each specific siRNA (Si).These knockdown studies revealed that PHLPP2 specifically activated AKT-p53-p21 signaling, whereas PHLPP1 did not affect this signaling (Fig. S3A).Cells with depletion of *** ** * PHLPP2 showed decreased cell proliferation and increased SAβ-Gal positivity, whereas such senescence traits were not in observed in PHLPP1-depleted cells (Fig. S3B, C).These results indicate that PHLPP2, but not PHLPP1, contributes to TRIM22mediated cellular senescence as a downstream regulator of TRIM22 and an upstream regulator of AKT.A previous study [33] reported that PHLPP2 expression is suppressed by Mut p53 in colorectal cancer.To investigate the expression of PHLPP2 in HCC cell lines, we analyzed the Cancer Cell Line Encyclopedia (CCLE)-Liver database, as well as conducted RT-qPCR and Western blot analyses.In the CCLE-Liver database, mRNA levels of PHLPP2 exhibited an increase in HCC cell lines with Mut p53 (SNU449, Huh7, and PLC/PRF/5) compared to those with Wt p53 (SK-Hep-1 and HepG2) (Fig. S4A).Subsequently, we performed RT-qPCR and Western blotting to assess PHLPP2 mRNA and protein levels in the HCC cell lines we used.RT-qPCR analysis indicated higher mRNA levels of PHLPP2 in the Mut p53 HCC cell lines (SNU449, Huh7, and PLC/PRF/5) compared to the Wt p53 HCC cell lines (SK-Hep-1 and HepG2) (Fig. S4B).Moreover, analysis of The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) database showed that the PHLPP2 expression was not lower in Mut p53 HCC tissues compared to Wt p53 HCC tissues (Fig. S4C).Western blot analysis revealed similar protein levels of PHLPP2 in the Mut p53 HCC cell lines compared to the Wt p53 HCC cell lines (Fig. S4D).In conclusion, our analysis suggests that the basal expression of PHLPP2 is independent with p53 status in HCC cell lines.

TRIM22 physically interacts with PHLPP2 and promotes its ubiquitin-mediated degradation
To examine whether TRIM22 directly regulates the level of PHLPP2, we used cycloheximide (CHX) to block de novo protein synthesis in TRIM22-overexpressed or IR-treated HepG2 cells.We found that TRIM22 overexpression significantly decreased the PHLPP2 protein levels in TRIM22-overexpressed and IR-treated cells (Fig. 3A, Fig. S5A, B).The PHLPP2 protein level in this system was rescued by the proteasome inhibitor, MG132, whereas the lysosome inhibitor, chloroquine (CQ), had no effect on this protein level (Fig. 3B, Fig. S5C).To assess whether TRIM22 regulates the PHLPP2 protein level through a physical association, we performed reciprocal immunoprecipitations (IP) in HepG2 cells transfected with TRIM22-Myc.TRIM22 and PHLPP2 could be reciprocally precipitated using either anti-Myc or anti-PHLPP2 in TRIM22-Myc-transfected cells, indicating that there is a physical interaction between TRIM22 and PHLPP2 (Fig. 3C).The cytoplasmic interaction between TRIM22 and PHLPP2 was further confirmed by the results of a proximity ligation assay (PLA) (Fig. 3D).Moreover, the interaction between TRIM22 and PHLPP2 was increased in IR-induced senescent HepG2 cells (Fig. S5D, E).
Together, these findings demonstrate that TRIM22 directly interacts with PHLPP2 and regulates its protein level to induce cellular senescence.
To elucidate the TRIM22 domain responsible for the interaction with PHLPP2, we generated Myc-tagged TRIM22 truncation mutants with deletions in the RING domain (ΔRING), BBox domain (ΔBBox), CC domain (ΔCC) or SPRY domain (ΔSPRY) (Fig. 3E, Upper).Co-IP and Western blot analyses demonstrated that the SPRY domain of TRIM22 was essential for interaction with PHLPP2, while the other domains (RING, BBox, and CC) did not influence the interaction with PHLPP2 (Fig. 3E, Bottom).Substrate phosphorylation can influence the substrate-E3 ligase interaction, leading to ubiquitination and degradation of the substrate [34].Review of the PhosphoSite database (www.phosphosite.org)revealed that PHLPP2 contains 10 phosphorylation sites at its C-terminal domain.To test whether phosphorylation of PHLPP2 might be responsible for its association with TRIM22, we generated PHLPP2 truncation mutants lacking the C-terminal domain (ΔCTD) (Fig. 3F, Upper).We found that PHLPP2 ΔCTD could not bind to TRIM22 (Fig. 3F, Bottom).This indicates that the SPRY domain of TRIM22 and the C-terminus of PHLPP2 are essential for the interaction between these proteins.Furthermore, TRIM22 Wt, ΔBBox, or ΔCC induced K48-linkage polyubiquitination of PHLPP2, whereas TRIM22 ΔRING or ΔSPRY had no effect on PHLPP2 ubiquitination (Fig. 3G).In IR-induced senescent HepG2 cells, the upregulation of TRIM22 expression increased the K48linked polyubiquitination levels of PHLPP2, whereas TRIM22 depletion decreased this parameter (Fig. S5F).TRIM22-induced the K48-linked polyubiquitination of PHLPP2 Wt, but not its C-terminal deleted mutant (PHLPP2 ΔCTD), which was validated using a K48-linkage specific polyubiquitin antibody (Fig. 3H).In TRIM22 Wt-overexpressed cells, the PHLPP2 protein level was decreased and AKT-p53-p21 signaling was activated, finally leading to cellular senescence (Fig. S6A-C).However, mutant TRIM22 lacking the RING-finger or SPRY domain failed to regulate the PHLPP2 protein level, activate downstream AKT-p53-p21 signaling, and induce cellular senescence (Fig. S6A-C).
These results demonstrate that TRIM22 physically interacts with PHLPP2 and promotes its ubiquitin-mediated proteasomal degradation, ultimately leading to cellular senescence due to AKT activation in HCC cells.
(Fig. 4B), indicating that the binding of PHLPP2 to TRIM22 was occurred though PHLPP2 phosphorylation per se.Thus, we investigated which kinase is responsible for PHLPP2 phosphorylation in TRIM22-overexpressed HCC cells.Our phosphoprotein array analysis revealed that TRIM22 overexpression led to the phosphorylation of 21 kinases (Fig. 4C).Using the STRING software with a confidence cutoff of 0.5, we predicted that AKT1, P70S6K, mTOR, and IKKα/β could potentially interact with PHLPP2 (Fig. 4D).Phosphorylation status of those kinases were confirmed by Western blot analysis of TRIM22-overexpressed cells (Fig. 4E).To further clarify the result, we knock downed AKT1, mTOR, P70S6K, IKKα, and IKKβ, and found that depletion of IKKα or IKKβ restored the PHLPP2 protein level in TRIM22-overexpressed cells (Fig. 4F).Moreover, IKKα and IKKβ are interacted with PHLPP2 in TRIM22overexpressed cells (Fig. 4G).
To explore the roles of IKKα and/or IKKβ in PHLPP2 phosphorylation, we performed PHLPP2 IP in IKKα-or IKKβ-depleted cells.We found that the interaction between PHLPP2 and TRIM22 was reduced in IKKβ-depleted cells in a PHLPP2 phosphorylation status-dependent fashion, but that this was not seen in IKKα-depleted cells (Fig. 5A).Similar results were obtained from TRIM22 IP assays in IKKα-or IKKβ-depleted cells (Fig. 5B).An in vitro kinase assay revealed that PHLPP2 was directly phosphorylated by IKKβ (Fig. 5C).Consistently, the degradation of PHLPP2 by TRIM22 was decreased in IKKβ-depleted cells (Fig. 5D) and TRIM22-induced K48-linked polyubiquitination of PHLPP2 was reduced by IKKβ knockdown (Fig. 5E).As the phosphorylation of IKKβ was increased in TRIM22-overexpressed HCC cells, we investigated the potential role of TRIM22 in regulating IKKβ phosphorylation.We observed that upregulation of TRIM22 increased the phosphorylation of IKKβ at Y188 in HCC cells with Wt p53, but not Mut p53 (Fig. S7A).Additionally, knockdown of TRIM22 in IR-treated HepG2 cells did not alter the phosphorylation level of IKKβ (Fig. S7B).Furthermore, TRIM22 overexpression in HepG2 cells dose-dependently induced the phosphorylation of IKKβ at Y188, in correlation with PHLPP2 degradation and AKT-p53 signaling activation (Fig. S7C).
In summary, our results indicate that TRIM22 induces the IKKβ-mediated phosphorylation of PHLPP2 and the subsequent degradation of PHLPP2 via ubiquitin-mediated proteasomal degradation, and that this involves a direct interaction between TRIM22 and PHLPP2.

TRIM22 expression is inversely correlated with PHLPP2 expression in HCC databases and patient specimens
To investigate the clinical significance of TRIM22 and PHLPP2 in HCC, we analyzed their expression levels in patients with HCC, as deposited to the International Cancer Genome Consortium Liver Cancer-RIKEN Japan (ICGC-LIRI-JP) (Fig. 6A) and TCGA-LIHC (Fig. 6B) databases.Analyses of these databases revealed that TRIM22 expression was downregulated in both total and paired HCC tumor tissues compared to normal tissues, while PHLPP2 expression was upregulated in HCC tumor tissues (Fig. 6A, B).Furthermore, our TCGA-LIHC database analysis showed that patients with high TRIM22 expression and low PHLPP2 expression had better overall survival (OS) rates (Fig. 6C).To support these findings, we collected 30 pairs of HCC and normal tissues and evaluated the protein levels of TRIM22 and PHLPP2.The results showed that TRIM22 protein levels were lower and PHLPP2 protein levels were higher in HCC tissues compared to normal tissues.Moreover, we observed that levels of phospho-IKKβ (Y188) in HCC tissues were lower than those in normal tissues (Fig. 6D).Furthermore, the IHC scores were negatively correlated between TRIM22 and PHLPP2 in both normal and tumor tissues (Fig. 6E).Taken together, our findings suggest that the expression levels of TRIM22 and PHLPP2 are inversely associated in HCC patient tissues.

DISCUSSION
The present study demonstrates that TRIM22 promotes HCC senescence by activating the AKT-p53-p21 signaling pathway.AKT, a serine and threonine kinase, is activated by phosphorylation at T308 or S473, and regulates cell survival, proliferation, growth, and glycogen metabolism [35,36].Our group and others reported that, although AKT is associated with tumor initiation and progression, its activation can induce cellular senescence in both non-transformed and cancer cells [37][38][39][40][41].This AKT-induced cellular senescence (AIS) can be triggered by various conditions, such as overexpression of myristoylated-AKT (Myr-AKT) and activation of receptors upstream of AKT [39].AIS can serve as a fail-safe mechanism against tumorigenesis.Our group and others also demonstrated that the loss of phosphatase and tensin homolog (PTEN), a major negative regulator of PI3K/AKT signaling, can induce cellular senescence in a p53-dependent manner that is called PTEN-induced cellular senescence (PICS) [37][38][39]42].These findings emphasize that AKT plays dual roles in critically regulating cell fate decisions between proliferation and senescence, depending on cellular context.
The induction of cellular senescence is an important anticancer strategy, as it can suppress tumor growth and enhance the vulnerability to combination treatments.Senescence can be triggered by various cancer therapies, particularly IR treatment.To identify E3 ligases that are involved in regulating therapyinduced senescence, we herein conducted expression profiling analysis of IR-treated senescent HepG2 cells.We found that TRIM22 is upregulated in response to IR-exposure in HCC cells.TRIM22 is an E3 ligase that can exhibit both tumor-promoting and tumor-suppressive roles in different cancers.Several TRIM22 isoforms play crucial roles in cancer biology.For instance, TRIM47 has been implicated in promoting tumor progression in colon and pancreatic cancer by degrading SMAD4 and FBP1 [43,44].In HCC, TRIM25 enhances tumor cell survival by targeting Keap1 for degradation [20].Conversely, TRIM7 and TRIM50 have been reported to suppress HCC progression by directly targeting Src and SNAIL for degradation, respectively [45,46].In colorectal cancer, TRIM67 functions as tumor suppressor by inducing p53induced apoptosis and inhibiting cell growth [47].TRIM22 is upregulated in glioblastoma (GBM) and promotes tumor growth and progression by modulating the stability of IKKγ and IkBα [29].
Conversely, TRIM22 is downregulated in osteosarcoma (OS) and gastric cancer [30,31].Overexpression of TRIM22 suppresses the proliferation and metastasis of OS cells by targeting NRF2 for degradation and activating the ROS/AMPK/mTOR/autophagy signaling pathway [31].In gastric cancer cells, TRIM22 inhibits cancer cell proliferation and migration by reducing the phosphorylation of SMAD2 [30].The present study demonstrates that TRIM22 critically contributes to the therapy-induced senescence of Immunoprecipitates were analyzed by Western blotting using the indicated antibodies.B IP was performed using anti-PHLPP2 antibody in TRIM22-overexpressed HepG2 cells.After IP, lysates were treated with λ-PPase and analyzed by Western blotting using the indicated antibodies.C Workflow of strategy used to identify kinase candidates that interact with and phosphorylate PHLPP2.D STRING analysis of PHLPP2-interacting kinases.E Western blot analysis of kinases in TRIM22-overexpressed HepG2 cells.F HepG2 cells were transfected with siRNA targeting each indicated kinase.After 48 hrs, the cells were harvested and analyzed by Western blotting using the indicated antibodies.G IP using anti-IKKα (left) or anti-IKKβ (right) in TRIM22-overexpressed HepG2 cells.Immunoprecipitates were analyzed by Western blotting using the indicated antibodies.
HCC cells.Our data indicated that TRIM22 functions as a tumor suppressor by directly regulating the level of PHLPP2.Previous studies have reported that TRIM22 induces apoptosis in osteosarcoma, monocyte, and neuron cells [31,48,49].However, in this study, we revealed that TRIM22 is a critical factor in cellular senescence in HCC cells.TRIM22 overexpression was shown to suppress cell proliferation by inducing cellular senescence without causing cell death in HCC.Our study showed consistent results from IR-induced and TRIM22-overexpressed senescent HCC cells, and from HCC patient tissues.p53 is a transcription factor that plays crucial roles in suppressing tumor growth by promoting cellular senescence, apoptosis, DNA repair, and other important processes.Mutations in p53 that result in the loss of its transcriptional activity can lead to cells taking on oncogenic functions, chemo-resistance, and other aspects of tumorigenesis [2,50,51].Approximately 70% of HCC patients having Wt p53 indicates that the frequency of p53 mutations is relatively low in HCC compared to other types of human cancer [50,51].The present study demonstrated that TRIM22, which is induced by Wt p53 under the IR-exposed condition, triggers HCC cell senescence by activating the AKT-p53-p21 signaling pathway.These findings indicate that Wt p53 is essential both upstream and downstream of TRIM22 for the induction of HCC cell senescence (Fig. 7).Mechanistically, TRIM22 degrades the AKT phosphatase, PHLPP2, to increase AKT phosphorylation in HCC cells.PHLPP2 belongs to the Pleckstrin Homology Domain Leucine-Rich Repeat Protein Phosphatase (PHLPP) family, the members of which negatively regulate PI3K/AKT signaling by dephosphorylating AKT at T308 and S473 [32].Moreover, Tantai et al. previously reported that TRIM46 activates AKT signaling by promoting the ubiquitination of PHLPP2 in lung adenocarcinoma (LUAD) [52].In this study, we found that TRIM22 overexpression phosphorylates PHLPP2, and this phosphorylation is crucial for the interaction of PHLPP2 with TRIM22.Unlike PHLPP2, we evidenced that the PHLPP family isoform, PHLPP1, failed to mediate senescence in this study.It is reported that TRIM22 overexpression increases the phosphorylation of IKKβ at S181 and Y188, resulting in IKKβ activation [53].Activated IKKβ induces the phosphorylation and subsequent degradation of substrates by recruiting E3 ligases [54,55].Consistent with these previous reports, we observed that PHLPP2 was phosphorylated by IKKβ activation in TRIM22-overexpressed cells, which is crucial for the recruitment of TRIM22.Furthermore, TRIM22 and IKKβ were negatively correlated with PHLPP2 in HCC patient samples.
Conclusively, we reveal a novel mechanism in which TRIM22 regulates PHLPP2 to promote HCC senescence.Targeting TRIM22 may present a promising therapeutic approach for the treatment of cancers, offering a new avenue for intervention in cancer therapy.

Transfection of siRNA and plasmids
Transfection of siRNAs (Bioneer, Daejeon Korea) and plasmids were performed using RNAi-MAX (Invitrogen, Carlsbad, CA, USA) and Lipofectamine 2000 (Invitrogen).Transfection medium were exchanged by regular growth media 6 hrs after transfection.The sequences of siRNAs used in this study were listed in Table S1.

Plasmid constructs
Full-length TRIM22 or TRIM22 mutants (TRIM22 ΔRING, ΔBBox, ΔCC or ΔSPRY) were obtained from pCMV6-TRIM22-Myc-DDK (RC207431, Origene, Rockville, MD, USA) and cloned into the pCMV-Myc vector.Full-length PHLPP2 was provided by Dr. KyeongJin Kim at Inha University, Korea and cloned into the p3xFlag vector.PHLPP2 ΔC-terminal (CTD) mutant were prepared based on p3xFlag-PHLPP2 Wt.All gene fragments were obtained by PCR amplification.The primers for plasmid construction used in this study were listed in Table S2.

Proximity ligation assay (PLA)
HepG2 cells seeded onto the glass coverslips were transfected with indicated plasmids or treated with IR.After 48 hrs, the cells were treated with 20 μM MG132 for 4 hrs.Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.1% Triton X-100 for 10 min and washed with DPBS.Protein-protein interactions were detected using Duolink ® PLA kit (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's instructions.The coverslips were mounted using Duolink ® In Situ Mounting Medium with DAPI (Sigma-Aldrich).Immunofluorescence was detected and visualized using Zeiss LSM510 confocal microscope.
RNA extract, reverse transcription and quantitative PCR (RT-qPCR) Total RNA was extracted from cells and tissues using TRIzol reagent (Molecular Research Center, Netherlands).RNA was reverse transcribed to cDNA using M-MLV reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instruction, and qPCR was performed using iQTM SYBR ® Green Supermix (BioRad Laboratories, Hercules, CA, USA) in a CFX ConnectTM RT-PCR Detection System (BioRad Laboratories).Housekeeping gene Actin was used as internal controls to normalize target mRNAs.The primer sequences for RT-qPCR were listed in Table S3.

Immunoprecipitation (IP) and Chromatin Immunoprecipitation (ChIP)
For IP, cells were lysed in NET-2 buffer (1 M Tris-HCl (pH 7.4), 5 M NaCl, 10% NP-40, 0.1 M PMSF and 0.2 M Benzamidine) containing 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate and 1 mM sodium orthovanadate.To remove non-specific bindings, cell lysates were pre-cleared using either protein A-Sepharose beads (GE Healthcare Bio-Science AB, Uppsala, Sweden) or G-Resin (GenScript, Piscataway, NJ, USA).Pre-cleared lysates were immunoprecipitated with specific antibodies or IgG for 4 hrs, followed by overnight incubation with protein A/G beads.The immunoprecipitants were washed with NET-2 buffer and eluted with 2× Laemmli sample buffer, followed by incubation at 95 °C for 10 min.For ChIP, IRtreated HepG2 cells were cross-linked with 1% formaldehyde for 5 min and stopped by 0.125 M glycine for 10 min at room temperature.The cells were washed with cold PBS, lysed with NET-2 buffer, and pre-cleared with protein A-Sepharose beads.The lysates were immunoprecipitated with either anti-p53 (Santa Cruz, FL-393) or rabbit IgG for 4 hrs, followed by incubation with protein A beads overnight.After washing with NET-2 buffer, DNAs bounds to proteins were purified by phenol-chloroform extraction and precipitated in ethanol.The DNA was then resuspended in DNase-and RNase-free water and analyzed by qPCR analysis.The proteins were detected using Western blot analysis.

Western blotting
The cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 2 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate) containing protease inhibitor (Roche) and phosphatase inhibitor (Sigma-Aldrich).The protein concentration of whole cell lysates was determined using Bradford Protein Assay (BioRad Laboratories).Equal amounts of proteins were mixed with 2× Laemmli sample buffer and incubated for 5 min at 95 °C.The proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK), and blocked with 5% Bovine Serum Albumin (BSA).The membranes were then incubated with the primary antibodies for overnight at 4 °C.After washing with 1× TBST, the membranes were incubated with an HRP-linked secondary antibody and signals were detected using Pierce™ ECL Western Blotting Substrate

Fig. 4
Fig.4PHLPP2 is regulated by IKKα and IKKβ in TRIM22-overexpressed cells.A IP using anti-PHLPP2 in TRIM22-overexpressed HepG2 cells.Immunoprecipitates were analyzed by Western blotting using the indicated antibodies.B IP was performed using anti-PHLPP2 antibody in TRIM22-overexpressed HepG2 cells.After IP, lysates were treated with λ-PPase and analyzed by Western blotting using the indicated antibodies.C Workflow of strategy used to identify kinase candidates that interact with and phosphorylate PHLPP2.D STRING analysis of PHLPP2-interacting kinases.E Western blot analysis of kinases in TRIM22-overexpressed HepG2 cells.F HepG2 cells were transfected with siRNA targeting each indicated kinase.After 48 hrs, the cells were harvested and analyzed by Western blotting using the indicated antibodies.G IP using anti-IKKα (left) or anti-IKKβ (right) in TRIM22-overexpressed HepG2 cells.Immunoprecipitates were analyzed by Western blotting using the indicated antibodies.

Fig. 5 Fig. 6
Fig. 5 Phosphorylation of PHLPP2 by IKKβ facilitates the interaction between PHLPP2 and TRIM22 and promotes the degradation of PHLPP2 by TRIM22.A, B IP using anti-PHLPP2 (A) or anti-Myc (B) was performed in IKKα-or IKKβ-depleted HepG2 cells following TRIM22 overexpression.Immunoprecipitates were analyzed by Western blotting using the indicated antibodies.C Western blotting of an in vitro kinase assay performed between IKKβ and PHLPP2.D Analysis of PHLPP2 protein stability.Con Si-or IKKβ Si-transfected HepG2 cells were transfected with EV or TRIM22-expressing vector.The cells were treated with cycloheximide (CHX) for the indicated times, harvested, and analyzed by Western blotting.Data are presented as mean ± SD (one-way ANOVA with Tukey's multiple comparison test, F(14,30) = 14.01, *P = 0.0153; # P > 0.9999; *P = 0.0437, n = 3).E Ubiquitination assay of PHLPP2 in IKKβ-depleted HepG2 cells following TRIM22 overexpression.

Fig. 7
Fig. 7 Schematic model proposed according to the findings of the present study.A proposed model for the function of TRIM22 in the degradation of PHLPP2 and the induction of cellular senescence in HCC cells (created with BioRender.com).